52 Anexo 1- Parecer do Comitê de Ética em Pesquisa da Unifesp
53 Anexo 2 - Termo de Consentimento
1 – Uso da reação em cadeia de polimerase e da eletroforese de campo pulsado para a caracterização microbiológica dos casos de infecção intraocular em um serviço de referência
2 – O objetivo deste estudo é usar a reação em cadeia de polimerase para a identificação mais precisa da infecção intraocular;
3 – Assim como em todos os casos suspeitos de infecção intraocular, será realizada injeção intraocular para retirada de amostras para identificação de micro-organismos causadores da doença, além da aplicação de antibióticos dentro olho;
4 – A injeção previamente descrita será feita sob anestesia local, com uso de colírios. O paciente poderá sentir leve incômodo durante sua realização. Assim como todo procedimento de injeção no olho, existe risco, apesar de pequeno, de descolamento de retina;
5 – Somente no final do estudo poderemos concluir a presença de algum benefício em acrescentar a reação em cadeia de polimerase na investigação das infecções intraoculares; 6 – Todos os procedimentos realizados para a investigação de infecção intraocular envolvem injeção no olho. Portanto, este estudo não aumenta os riscos além dos já existentes nesses casos;
7 – Garantia de acesso: em qualquer etapa do estudo, você terá acesso aos profissionais responsáveis pela pesquisa para esclarecimento de eventuais dúvidas. O principal investigador é o Dr Gustavo Barreto de Melo que pode ser encontrado no endereço Rua Botucatu, 822 Telefone(s) 5085-2000. Se você tiver alguma consideração ou dúvida sobre a ética da pesquisa, entre em contato com o Comitê de Ética em Pesquisa (CEP) – Rua Botucatu, 572 – 1º andar – cj 14, 5571-1062, FAX: 5539-7162 – E-mail: [email protected]
8 – É garantida a liberdade da retirada de consentimento a qualquer momento e deixar de participar do estudo, sem qualquer prejuízo à continuidade de seu tratamento na Instituição; 9 – Direito de confidencialidade – As informações obtidas serão analisadas em conjunto com outros pacientes, não sendo divulgado a identificação de nenhum paciente;
10 – Direito de ser mantido atualizado sobre os resultados parciais das pesquisas, quando em estudos abertos, ou de resultados que sejam do conhecimento dos pesquisadores;
11 – Despesas e compensações: não há despesas pessoais para o participante em qualquer fase do estudo, incluindo exames e consultas. Também não há compensação financeira relacionada à sua participação. Se existir qualquer despesa adicional, ela será absorvida pelo orçamento da pesquisa.
54 propostos neste estudo (nexo causal comprovado), o participante tem direito a tratamento médico na Instituição, bem como às indenizações legalmente estabelecidas.
13 - Compromisso do pesquisador de utilizar os dados e o material coletado somente para esta pesquisa.
Acredito ter sido suficientemente informado a respeito das informações que li ou que foram lidas para mim, descrevendo o estudo “Uso da reação em cadeia de polimerase e da eletroforese de campo pulsado para a caracterização microbiológica dos casos de infecção intraocular em um serviço de referência.”
Eu discuti com o Dr. Gustavo Barreto de Melo sobre a minha decisão em participar nesse estudo. Ficaram claros para mim quais são os propósitos do estudo, os procedimentos a serem realizados, seus desconfortos e riscos, as garantias de confidencialidade e de esclarecimentos permanentes. Ficou claro também que minha participação é isenta de despesas e que tenho garantia do acesso a tratamento hospitalar quando necessário. Concordo voluntariamente em participar deste estudo e poderei retirar o meu consentimento a qualquer momento, antes ou durante o mesmo, sem penalidades ou prejuízo ou perda de qualquer benefício que eu possa ter adquirido, ou no meu atendimento neste Serviço.
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Assinatura do paciente/representante legal Data / /
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Assinatura da testemunha Data / /
para casos de pacientes menores de 18 anos, analfabetos, semi-analfabetos ou portadores de deficiência auditiva ou visual.(Somente para o responsável do projeto)
Declaro que obtive de forma apropriada e voluntária o Consentimento Livre e Esclarecido deste paciente ou representante legal para a participação neste estudo.
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55 Anexo 3 - Artigo relacionado publicado como coautor
Bispo PJ, Melo GB, Höfling-Lima AL, Pignatari AC. Detection and Gram
Discrimination of Bacterial Pathogens from Aqueous and Vitreous Humor Using Real-Time PCR Assays. Invest Ophthalmol Vis Sci. 2010 Aug 11. [Epub ahead of print]
Abstract
Purpose. To develop and apply real-time PCR protocols to detect and classify the Gram status of bacterial pathogens in aqueous and vitreous humor collected from clinically suspected intraocular infections. Methods. The analytical specificity of two PCR assays, SYBR Green 16S rDNA-Based Universal PCR (SGRU-PCR) and a Multiplex Gram-Specific TaqMan-Based PCR (MGST-PCR), was determined using 31 clinically important pathogens, including 20 Gram-positive and 11 Gram-negative. Analytical sensitivity was determined using a 10-fold dilution of S. epidermidis and E. coli DNA. Assays were further tested on aqueous (n=10) and vitreous humor (n=11) samples collected from patients with a clinical diagnosis of intraocular infections. Results. DNA was amplified from all control bacterial isolates using SGRUPCR. MGST-PCR correctly classified the Gram status of all these isolates. The limit of detection of S. epidermidis and E. coli DNA was 100 fg/microl using SGRUPCR (E = 0.82 and 0.86; r2 = 0.99) and 1 pg/microl for MGST-PCR (E = 0.66 and 0.77; r2 = 0.99. For clinical intraocular samples, positivity of culture was 47.6% and for real-time PCR assays 95.2%. Gram classification was achieved in 100% of PCR-positive samples using MGST-PCR. Among microbiologically negative samples, real- time PCR assays were positive in 90% of cases. False positive rate in control aqueous was 3.2% and control samples of vitreous were negative. Conclusions. Real-time PCR assays demonstrated a good correlation with culture-proven results. With the use of these methods, bacterial detection was improved from 47.6% to 95.3%, demonstrating to be sensible and fast tests for bacterial endophthalmitis diagnosis.
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ABSTRACT
Purpose: I- To report the incidence of bacterial endophthalmitis and the frequency of its causative microorganisms at a university-setting in Brazil. II- To assess the distribution of microorganisms isolated from patients with bacterial endophthalmitis and their antimicrobial susceptibility. III- To determine the usefulness of real-time polymerase chain reaction (PCR) assays in the diagnosis of post-operative bacterial endophthalmitis in clinically diagnosed infectious cases and to test for bacterial DNA in control samples.
Methods: I- Main data assessed from cases of presumed postoperative bacterial endophthalmitis from 2002 to 2008 were: number of cataract surgeries performed, incidence of endophthalmitis, positivity of Gram staining and culture (aqueous and/or vitreous), and antimicrobial susceptibility. II- Retrospective analysis of medical and microbiological records of patients with suspected diagnosis of endophthalmitis was carried out. The following information was assessed: number of clinically diagnosed and culture-positive endophthalmitis cases, predisposing factors for the infection, Gram staining and culture outcomes (aqueous and/or vitreous), microbial characterization and frequency, and antimicrobial susceptibility. III- Samples from eyes of patients with clinically diagnosed infectious endophthalmitis after cataract surgery were included, as well as vitreous and aqueous samples from eyes without infection or inflammatory reaction. Universal and Gram-specific real-time PCR were carried out and the technique’s sensitivity and cycle thresholds were determined. Gram staining and culture were also assessed.
Results: I- Seventy-three eyes of 73 patients (43 females and 30 males) developed presumed infectious endophthalmitis after 24,590 cataract surgeries. The incidence decreased from 0.49% in 2003 to 0.17% in 2006 and stabilized afterwards. Coagulase negative Staphylococci (CoNS) and Streptococcus viridans (56.5% and 15%, respectively) were the most common bacteria. Culture and Gram stain were negative in 36.9%. CoNS presented susceptibility rates of 80%-sensitivity to oxacillin, 90% to fourth-generation quinolones and 100% to vancomycin. II- A hundred and seven (46%) of 231 patients with bacterial endophthalmitis showed positive results by gram-stain or culture. Of these, 97 (42%) patients were positive for culture only. Most of them (62%) were secondary to a surgical procedure (postoperative), 12% were post-traumatic and 26% were secondary to an unknown source. A total of 100 microorganisms were isolated (38 aqueous and 67 vitreous samples) from the 97 culture-positive cases. Coagulase-negative Staphylococcus (CoNS) (48%) were most frequently isolated, followed by Streptococcus viridans (18%) and Staphylococcus aureus (13%). Antimicrobial susceptibility for CoNS was as follows: amikacin - 91.6%, cephalothin - 97.9%, ceftriaxone - 50%, ciprofloxacin - 62.5%, chloramphenicol - 91.8%, gatifloxacin - 79.5%, gentamicin - 72.9%, moxifloxacin - 89.5%, ofloxacin - 70.8%, oxacillin - 58.3%, penicillin - 33.3%, tobramycin - 85.4%, and vancomycin - 100%. III- Eleven patients with infectious endophthalmitis (9 vitreous and 7 aqueous samples) after cataract surgery were included, as well as 12 vitreous and 50 aqueous samples from control eyes. It was possible to identify 80% and 75% of the patients with infectious endophthalmitis by Gram staining and culture, respectively. Real-time PCR assays were positive in 91% of the patients, using either aqueous and/or vitreous samples. None of the 12 vitreous controls were positive and two of the aqueous control samples were positive by real-time PCR. The Ct cutoff value for universal-PCR was 36 (sensitivity: 93.8%; specificity: 100%) and 38 for Gram-specific-PCR (sensitivity: 93.8%; specificity: 100%). Gram-positive microorganisms prevailed and visual acuity varied according to the causative bacteria. Conclusions: I- The rate of bacterial endophthalmitis, microbial isolates and antibiotic susceptibility are in accordance with
the literature. Despite using prophylactic antibiotic drops, organisms from infected cases susceptible to the antibiotics topically applied were found. II- Gram-positive bacteria were the major causes of infectious endophthalmitis. CoNS was the most common isolate. Susceptibility to oxacillin and fourth-generation quinolones was lower than previously published. III- Real-time PCR is a fast and sensitive diagnostic tool in cases of bacterial endophthalmitis. As a quantitative technique, it may also serve as a new and unique application: the distinction between contamination and infection based on the cycle threshold value.
Keywords: endophthalmitis; epidemiology; diagnosis; polymerase chain reaction; microbial sensitivity tests.